This invention relates to the purification of lipoproteins to produce a material high in cholesterol content and to the use of such high-cholesterol material as a cholesterol reference standard for determining the cholesterol content of a body fluid.
Determination of serum cholesterol levels is of importance because elevated levels may be useful in the diagnosis of certain maladies. For example, determination of serum cholesterol is used to screen for coronary artery diseases, diabetes mellitus, nephrosis, hepatic and thyroid diseases and metabolic disorders caused by endocrine disturbances.
Clinical chemistry procedures have been developed to enable the determination of cholesterol levels in serum. These procedures generally involve the use of cholesterol reference standards which include human serum and organic salts as components. U.S. Pat. No. 4,045,176 describes the preparation of a cholesterol control standard by adding either higher density human lipoproteins or isolated non-primate lipoproteins (e.g., bovine) to human serum.
U.S. Pat. No. 3,764,556 describes a method for preparing a lipid control sample by isolating a lipid-rich fraction of human plasma and adding equine, bovine or human serum.
U.S. Pat. No. 3,260,648 describes a composition for cholesterol determination which is lyophilized human blood plasma containing an organic solubilizing agent.
Because control standards have been thought to be more reliable if the physical and physiological make-up of the control closely parallels that of the substance to be tested, heretofore cholesterol reference materials have been based on the use of human serum or plasma which have been "adjusted" with organic salts, solubilizing agents or equine, bovine or human serum, as described in the above patents.
Such procedures involving the use of human serum or plasma suffer several disadvantages. First, the presence of human serum or plasma involves a danger to the user due to the possibility of hepatitus virus being present in the serum. Second, the presence of human serum or plasma increases the costs of such a cholesterol control standard.
There is thus a need for a cholesterol reference material that is not used in conjunction with human serum or plasma. The lipoprotein cholesterol of the present invention is suitable for use as a cholesterol reference material in determining the cholesterol content of human serum or plasma, without the presence of human serum or plasma.
Various methods have been used in the art to purify lipoproteins. The most widely used method is ultracentrifugation wherein the lipoprotein classes are separated from the bulk of plasma proteins and from each other by differences in density.
Another purification method involves precipitation and extraction. For example, U.S. Pat. No. 4,104,286 describes isolation of cholesterol from whole egg and dried egg yolk. The patentee describes extraction with ethanol, saponification in aqueous alkali hydroxide and concentration and purification with a hydrocarbon solvent in methanol. Other precipitation methods involve the use of sulfated polysaccharides combined with an alkaline earth. These reagents are relatively specific for lipoprotein precipitation.
A third method involves the adsorption of lipoproteins on inorganic matrices. In contrast to the ultracentrifugation and precipitation and extraction methods where the lipoproteins are purified and recovered, adsorption methods are generally used to remove lipoproteins from serum or plasma; the lipoprotein is then discarded as an impurity. U.S. Pat. No. 4,081,431 describes blood separation wherein a Factor VIII-enriched protein is obtained by removing the lipoprotein-containing Factors II, VII, IX and X as impurities by treatment with silica as a solid adsorbant.
The major stumbling block in using adsorption technology for purification of lipoprotein complexes is the difficulty of recovering the lipoprotein.
The most relevant prior art located appears to be an article in Zeit. Klin. Chem. 6 (3) 186-190 (May 1968).
The authors W. Stephan and L. Roka, describe the removal of .alpha..sub.1, .alpha..sub.2 and .beta.-lipoproteins from human sera. The method involves adsorbing human sera on colloidal silicic acid. The adsorbed proteins are then centrifuged and frozen, thawed and extracted by the use of a high salt concentration at a pH of 9. According to the authors, recovery of the maximum amount of adsorbed protein occurs at a pH of 9.0 and a salt concentration of 12 percent NaCl weight/volume. The authors disclose that the recovery of the adsorbed protein decreases drastically if the salt concentration varies from a concentration of 12 percent. (This is equivalent to a salt concentration of approximately 2.0 M).
None of the prior art patents, or the article discussed above teaches or suggests the improved results obtained by eluting an adsorbed lipoprotein complex at a pH of 10 to 11.5, adjusting the salt concentration to about less than 0.05 M, heating the eluted lipoprotein, adding an alkaline carbonate and alkaline earth salt, and adjusting the pH from about 6.5 to 9.0. None of the prior art referred to suggests the use of a cholesterol reference material produced as described from plasma or serum which is not used in conjunction with organic salts, solubilizing agents or human plasma or serum.